In this study, we investigated the Stabilization of bitumen emulsions by scleroglucan, a rigid triple-helix forming biopolymer, in combination with a pH-sensitive cationic surfactant. Various aspects of the emulsification process and the final composition influence the Stabilization. We examined two different methods to add scleroglucan to the emulsion: either by adding it to the aqueous surfactant solution before emulsification, denoted ‘pre-emulsification addition’ (pre-EA), or by addition to the emulsion after emulsification (post-EA). We investigated scleroglucan concentrations in the aqueous phase ranging between 0.017 and 0.07 w/w%. The emulsions were evaluated according to the European EN 13808 standard used for cationic bituminous emulsions, as well as by rheological analysis. We observed an improvement of the storage stability upon pre-EA at a biopolymer concentration as low as 0.017 w/w% in combination with an increased particle size, whereas the breaking index (characterising breaking of the emulsion in presence of ‘aggregates’ = stones) was not influenced. The rheological data show a minor viscosity increase by scleroglucan in the pre-EA formulation at low scleroglucan concentrations (0.017–0.05 wt.%) where Stabilization already improved dramatically. This indicates that the stabilization mechanism is not only governed by a viscosity increase but also by interfacial stabilisation effects were polymer is adsorbed onto the adsorbed surfactant. In a separate experiment we changed the conformation of scleroglucan by subjecting it to extreme pH values and by dissolution in DMSO, in order to study the role of the triple helix conformation in the stabilization mechanism. Scleroglucan becomes less effective in a denatured and hydrolysed state confirming the crucial role of the triple helix conformation in the Stabilization of bitumen emulsions.